Platelet- and plasma-derived factor Va serve an essential role in thrombin generation catalyzed by the prothrombinase complex, a 1:1, Ca2+-dependent complex of the cofactor factor Va and the serine protease factor Xa bound to activated platelets adhered to sites of vascular injury. Recently, our laboratory demonstrated that the entire pool of the platelet-derived procofactor, factor V, originates from plasma through endocytosis of factor V by platelet precursors, megakaryocytes, via a two receptor system consisting of a specific, as of yet, unidentified factor V receptor and LDL receptor-related protein-1 (LRP-1). In this model, it is hypothesized that factor V binding to its specific receptor facilitates the binding of another factor V molecule to LRP-1, which then mediates its endocytosis. This system represents a unique function for LRP-1 whereby endocytosed factor V is modified to yield a functionally distinct molecule, and not destined for degradation. The major goal of this project is to continue to characterize this two-receptor system for factor V endocytosis by megakaryocytes. CMK and CD34+ ex vivo-derived megakaryocytes, isolated and separated based upon their ability to endocytose factor V, will be used in several complementary approaches including co-immunprecipitation and state-of-the-art mass spectrometric techniques to identify the specific, factor V receptor. Once identified, anti-factor V receptor antibodies will be produced and used in combination with anti-LRP-1 antibodies to delineate the fate of these receptors subsequent to factor V endocytosis. Receptor expression as a function of megakaryocyte differentiation will also be determined as the ability of megakaryocytes to endocytose factor V occurs early in their development, and is lost in the mature platelet. In other studies, the ability of factor VIII, a protein highly homologous to factor V whose clearence is mediated by LRP-1, to interact with this receptor system on ex vivo-derived megakaryocytes will be determined. Domains important for this interaction will be defined and used to synthesize complementary factqr V peptides in order to identify the factor V domains important for its interactions with both receptors. As the two receptor model suggests that receptor-receptor interactions are necessary for factor V endocytosis, and LRP-1 phosphorylation regulates its function, the phosphorylation state of serine/threonine residues in the cytoplasmic tail of LRP-1 will be correlated with factor V binding and endocytosis. Since platelet-derived factor Va plays a more important role in maintaining normal hemostasis than does its plasma counterpart, and is physically and functionally distinct, these studies will increase our understanding of how megakaryocytes acquire, process and package this critical coagulation factor. Successful completion of these goals will help to define the cell membrane events that regulate acquisition of factor V by megakaryocytes all of which will be potential therapeutic targets in hypercoagulable and thrombotic states. (End of Abstract)